The invention relates to a counting compartment for accommodating a sample of biological material consisting of microscopic particles in a liquid medium for the quantitative microscopic examination of this material.
When examining body fluids, it is often desirable to study samples of such biological material under the microscope over a certain time. When examining sperm, for example, the aim is to establish how many spermatozoa are present in the sample and also how motile they are.
To carry out this investigation, a sample of a certain thickness is to be subjected to microscopic examination in a counting compartment, using a grid built into the eyelens of the microscope. Such a grid may be divided into a hundred squares, and the number of spermatozoa in each of a representative number of squares can be counted by the investigator in order to determine the total number of spermatozoa in the whole grid area. Such a grid may also be provided in the counting compartment. The number of spermatozoa in a square can be e.g. about one hundred to twohundred.
From U.S. Pat. Nos. 4,911,782 and 5,200,152 a method is known for conducting such determinations with the aid of a counting compartment formed by two transparent plates joined together by a connecting layer composed of a cured plastic.
However, the manufacture of such a counting compartment is complicated and expensive, because it involves the use of a photo-resist method for applying a plastic as the desired boundary of the compartment.
The aim of the present invention is to provide a counting compartment and a manufacturing method for it, which do not have this shortcoming and do not involve the use of the photo-resist method.
This aim is achieved according to the present invention by a counting compartment of the type indicated above, which is characterized in that the connecting layer contains material particles which are separate from one another and have a size which determines the depth of the counting compartment, which material particles are essentially in contact with the two plates.
The counting compartment can easily be constructed by using material particles which have a size which determines the depth of the counting compartment; furthermore, counting compartments with depths suitable for different biological materials can be produced by using material particles of appropriate dimensions.
The fact is that the examination of a sperm sample calls for a counting compartment with a depth of 12-20 microns, while the examination of blood cells needs a counting compartment with a depth of only 8-12 microns. Counting compartments with the required depth can be made by choosing material particles of the right size from amongst material particles measuring between 6 and 40 microns.
The particles which determine the depth of the counting compartment lie at a regular distance from one another in order to ensure a uniform depth for the counting compartment over its entire surface. Preferably, these particles, when their masses are equal, are at equal distances from one another. Furthermore, and preferably, these particles are substantially completely surrounded by the solid material of the connecting layer, formed from a fluent material.
To prevent clotting of these material particles, the particulate material chosen is preferably a non-magnetic, electrically highly resistant substance and, more preferably, aluminium oxide, but other materials and even plastic particles can also be used.
To prevent escaping, by capillary action, and evaporation of liquid from the sample of biological material consisting of microscopic particles in the counting compartment, the solid material of the connecting layer extends all round and up to the free end of the outlet of the compartment. This minimizes evaporation, so that no streaming occurs in the sample housed in the counting compartment.
The top and bottom plate are preferably both made of a material which transmits UV and/or visible light, preferably glass, and the top plate should be selected to be as thin as possible in order to facilitate the examination by means of a microscope.
The invention also relates to a method for the manufacturing of a counting compartment to accommodate a sample of biological material consisting of microscopic particles in a liquid medium for the quantitative microscopic examination of this material, which counting compartment comprises two transparent plates held at a fixed distance from each other and joined together by a connecting layer, and at least one counting compartment situated between the plates, bounded by the connecting layer and fitted with an inlet and an outlet. According to the invention, this method is characterized in that a fluent mixture of mutually repelling material particles and a liquid adhesive is applied to one of the plates, the other plate is placed on this fluent mixture, and pressure is exerted thereon until the two plates are essentially in contact with the largest material particles in the fluent material, which largest material particles determine the depth of the counting compartment.
It will be obvious that by using the above liquid mixture, one can easily apply this mixture to one of the plates, and the other plate can then be pressed on to this liquid material until the two plates are in contact with the largest particles present in the fluent mixture.
The mutually repelling material particles preferably carry an electrostatic charge and consist of a non-magnetic, electrically strongly insulating, material and are then charged electrostatically.
By using particles carrying an electrostatic charge, the particles will always lie at the same distance from one another, this distance depending on the size of the particle and on the charge.
A counting compartment according to the invention can be used to carry out biological determinations on samples of sperm, of blood and of malignant tissue, such as cancer.
From U.S. Pat. No. 4,689,307 it is known to provide microbeads as spacers between microscope slide and cover-slip around a sample for microscopic investigation. They can be added in advance to an aqueous liquid.
From EP-A 0 321 889 it is known to provide microbeads onto a microscope slide by fixing them in advance in a solid residue of dried liquid adhesive. This adhesive is soluble in the sample to be examined.
Neither one of these publications discloses the use of microparticles in a solid connecting layer for the formation of a counting compartment. Furthermore, there is no indication for the manner in which microspheres or other material particles which determine the spacing can be added without clotting to a viscous mass which, after curing, will not dissolve into the sample under examination.